Project Summary tRNA fragments (tRFs) defend the host against mobile genetic elements and function in RNA silencing. We found that 3'-derived tRNA fragments (3'-tRFs) inhibit long terminal repeat (LTR)-retroelements, which use the 3'-end of tRNAs to prime reverse transcription at their highly conserved primer binding site (PBS). 3'-tRFs are highly expressed in cancer and stem cells supporting our central hypothesis that 3'-tRFs protect the genome from transposon damage during epigenetic reprogramming in development and disease. Our rationale is that 3'- tRF mediated inhibition of LTR-retroelements is highly conserved and is an ancient link between transposons, RNA interference (RNAi), and genome stability. The overarching goal of this proposal is to determine key factors involved in this novel small RNA silencing mechanism and how they intersect with the RNAi pathway. First, we will develop reporter assays for the most highly active endogenous retroviruses (ERVs) in the mouse, IAP and ETn/MusD, which will resolve the timing of retrotransposition and be suitable for screening for proteins that mediate silencing by tRFs. We hypothesize that 3'-tRFs protect the preimplantation embryo in the absence of epigenetic repression of ERVs, similar to piRNAs in the germline, and we will test the in vivo impact of tRFs in mouse preimplantation embryos after pronuclear injection of luminescence-based ERV reporters. To dissect the role of RNAi proteins in 3'-tRF silencing, we will determine tRF expression, subcellular localization, retrotransposition rates, and diagnostic retroviral intermediates during candidate knock-down and knock-out. Catalytically deficient RNAi mutants will help resolve the role of these enzymes in 3'-tRF biogenesis versus binding and target recognition. Lastly, we will use RNA-protein pull-downs and a targeted CRISPR knock-out screen that scores for retrotransposition to identify novel RNA-binding proteins that function in 3'-tRF silencing of mobile elements. Repressive chromatin marks and full-length tRNA levels in the same cells will complete the picture. 3'-tRF biogenesis and silencing are at the root of a cellular decision whether to keep tRNAs for translation - and retroelement replication - or produce tRFs to inhibit them. Expression of LTR-retroelements not only threaten genome stability, but murine and human ERVs that are targeted by 3'-tRFs are also essential for stem cell pluripotency. Therefore, it is of high priority to determine the mechanism and scope of retroelement regulation by tRFs. Targeting of the PBS by 3'-tRFs is a unique vulnerability of this highly abundant and dispersed class of mobile elements and might enable innovative approaches towards treatment of infectious LTR-retroviruses, such as HIV. Understanding to what extent tRNAs are a substrate of the RNAi pathway will add novel perspective to the arms race between mobile elements and their hosts.